Deinococcus radiodurans, known for being the most radioresistant organism, is a good candidate for use in the remediation of radioactive industrial waste sites due to (a) its extraordinary capacity to thrive even in the presence of high levels of radiation and (b) the high level of genome plasticity shown in some strains that introduces the possibility for D. radiodurans to maintain, replicate, and express large fragments of foreign DNA. This pilot study will show the ability of D. radiodurans to remediate mercury ion contaminated soil in the areas immediately surrounding radioactive waste sites. This method is designed for initial application on Orchid Island, Taiwan, the major site for the storage of Taiwan's mid- to low-level radioactive wastes, and eventually to the storage sites of said wastes after they are transported to sub-standard waste storage facilities in Pyungsan, North Korea.

Introduction

Biological problem: The storage of radioactive wastes is a delicate matter. Some of the wealthiest and most technologically advanced nations in the world still have difficulty packaging and storing their radioactive wastes in a responsible manner that is effective on a sufficiently long term scale. This issue of maintaining safe storage facilities for long enough periods of time is of particular concern when considering the storage of radioactive wastes with half lives on the order of thousands of years. The methods for storing radioactive waste substances range from above-ground storage in metal containers to the subterranean storage in concrete blocks seen in the present situation between Taiwan and the Democratic Peoples Republic of Korea (DPRK). Underground storage facilities present very real threats that could lead to drastic changes in the lives of all who depend on water in the region. These include soil contamination and, subsequently, the potential for large-scale destruction of ecological systems, ranging from the organisms inhabiting the immediate niche to contamination of the water table. Some of the most noteworthy effects of exposure to radioactive wastes stem from DNA damage that manifests itself in humans in the form of increased occurrences of cancer and birth defects. Industrial waste sites, such as the one presently being studied on Orchid Island, consist of a diverse mix of waste products from industry, including used contamination suits, research wastes from hospitals and universities, and other rubbish, all of which are encased in blocks of concrete (The Economist 1997b). Often, the only common thread that unites the wastes is that they are contaminated with radioactivity.

The state-run Taiwanese power company, Taipower, is responsible for the storage of radioactive wastes from the country's three reactors and from a number of medical and research facilities. Taipower signed an agreement with the North Korean government in 1997, in which Taipower would pay approximately US$1,200 per barrel for the shipment and, storage of 100,000-200,000 barrels of low-level radioactive wastes (Gyorgy 1997; The Economist 1997a). An effort of this type and magnitude was unprecedented. It will involve the transport of thousands of tons of radioactive waste across the Yellow Sea to be sequestered in a third world country, in abandoned coal mines that sit atop major tectonic fault lines. These types of waste sequestration prove to be extremely difficult and technical projects even under the most favorable conditions. For the DPRK, many fear that the risks for contamination by the leakage of heavy metals and other toxic and radioactive wastes into the soil are particularly great.

Deinococcus radiodurans (Gr. adj. deinos strange/unusual; coccus grain or berry; radiodurans radiation tolerant) is a gram positive, red-pigmented, non-motile, aerobically respiring, coccus (0.5-3.5µm) that appears in pairs or tetrads (Murray 1981). Although officially designated gram positive, D. radiodurans has a plasma membrane as well as an outer membrane that are thought to be an artifact of recent common heritage with gram-negative relatives (Mrázek 2002). Optimal growth occurs at 25°C - 35°C and no resting phase cell type has been observed. D. radiodurans is most noted for its unmatched capacity to withstand large acute doses of ionizing and ultraviolet radiation (1,500 kilorads), as well an ability to survive, apparently unharmed, exposure to sustained, high levels of radiation (6 kilorads, approximately 10 times the lethal dose in humans) (Makarova 2001). From an evolutionary standpoint, this extraordinary resistance is curious given the absence of significantly high radioactive sites on the planet. This observation has led to the hypothesis that D. radiodurans' radioresistance is a byproduct of its extreme desiccation resistance, as it has been shown to survive even after 6 months on a glass slide maintained in a desiccator (Minton 1996). Another theory holds that D. radiodurans' radioresistance developed as a fortuitous consequence of its capacity to endure large-scale DNA damage. Although the natural niche of this bacterium has not been characterized thus far, D. radiodurans has been isolated from a variety of sites including meat packaging plants, sawdust, fecal matter, streams, and waterways (Makarova et al. 2001).

Population/Country: This study will take place with two distinct peoples and locations in mind: the Yami of Orchid Island and the Koreans of the DPRK. Due to the far-reaching consequences and sensitive nature of the topic of waste disposal, it is important to have an understanding of the cultural and historic context of each of the two populations in the focus of this study.

At just over 85,000 square miles (about the size of New York and Pennsylvania combined) on the coast of northeast Asia, the Korean peninsula is an area that has consistently suffered due to its strategic location. Wedged in-between three great and historically volatile nations, Russia, Japan and China, it has historically held enormous tactical importance. The people of the Korean peninsula have endured approximately 900 invasions in just over 2000 years of recorded history, including five major periods of foreign occupation by China, the Mongols, Japan, the Soviet Union, and the United States (Oberdorfer 2001).

Today, Korea exists as one of the most volatile and closed regions in the world. The peninsula has been divided along the 38th parallel for nearly 60 years. The line that separates the two Koreas serves as one of the starkest juxtapositions in the world. South of the 38th parallel exists a bustling, vibrant, and stable nation that is growing and flourishing as a member of the international community. To the north lies a nation reeling under the effects of the devastating 50-year reign of Kim Il Sung that is marked by economic mismanagement, a cult of personality, and the construction of a vast and terrible military force. After the recent death of Kim Il Sung, the nation has improved little, suffering 3 million deaths from starvation between 1996-2000 (Johnstone 2000; Reltien 2001), devastation as a result of natural disasters, low agricultural yields, and a national infrastructure that is insufficient to provide consistent basic services such as hospitals or heat even in the most developed regions. The sheer scale of the mismanagement, coupled with a disregard for basic human rights is an extraordinary tragedy. As an example: in 1999, the North Korean food deficit was estimated at 1.3 million tons. In the same year, over 1 million tons of food aid was sent to the DPRK yet the nation persisted in harboring one of the more significant famines that the world had seen in years (Action Against Hunger 2000). Given the historical context and the dire state of the nation, the international community is understandably dubious that the North Korean government will spend the $230 million dollars of revenue from the waste transfer deal on the proper transport and storage of the wastes (The Economist 1997a).

Orchid Island is the location of the Taipower radioactive waste site that will be relocated to the DPRK if the aforementioned deal proceeds as planned. Long before the Taiwanese government designated the island as a waste site, it was home to a small indigenous tribe called the Yami, who have been a vocal group in resisting the expansion of the waste storage facility. The Yami are an aboriginal people of Lanyu (Orchid Island), and number just under 3000 worldwide, with approximately 2000 living on the island. They are a people distinct in culture and language from other Taiwanese and from mainland Chinese through a long history of isolation. With no significant trade, the Yami have a largely subsistence culture that lives by cultivating taro and sweet potatoes, raising pigs, and fishing. There is no written form of the Yami language. In recent years, tourism has played a major part in the Yami economy as mainlanders and other travelers visit the small island 62 km from mainland Taiwan to enjoy the beauty of the island and to interact with the native people. The Yami are a stalwart people who make organized and clear demands to the Taiwanese governments. In one standoff, the Yami threatened to dig up the barrels and throw them into the sea unless the wastes were removed from the island in order to attract international attention to their plight. Thus, it is not difficult to appreciate the delicate conflict that rests in the hands of the Taiwanese officials concerning the matter of waste disposal. The natives of Orchid Island are proving to be less naïve and docile than the government had anticipated and the international uproar in response to the proposed waste transfer plan was enormous and effective in slowing the process (Asia 1997).

Proposed research

Deinococcus radiodurans is unique among the deinobacteria for its pliability with regard to genetic manipulations. This is most likely a result of the high transformability of its chromosomal and plasmid DNA (Brim et al. 2000; Makarova 2001). The well-defined E. coli metal resistance gene, merA, will be cloned into D. radiodurans with a two-fold purpose: First, the gene will confer general resistance to the harmful effects of some of the most common heavy metal wastes. Second, it will enable D. radiodurans to metabolize some of the metal components into less toxic, insoluble forms. MerA encodes mercuric ion reductase, MerA, which converts highly toxic, thiol-reactive mercuric ions Hg(II) into the nearly inert, monoatomic, volatile Hg(0). Thus, the merA+ D. radiodurans will serve as a doubly armored waste disposal agent due to its inherent ability to resist radiation and its manufactured ability to resist the damaging effects of high oxidation state metals.

This project will serve as a pilot study of the ability of merA+ D. radiodurans to remediate soil contaminated with a mixture of radioactive and other toxic wastes. As previously stated, merA+ D. radiodurans will be introduced to soil in a model that parallels the Orchid Island radioactive waste storage sites. This pilot study will serve the purpose of testing the effectiveness of the method in a relatively stable situation in order to be prepared for the uprooting, transport, and storage of potentially hundreds of thousands of barrels of wastes in the DPRK that will undoubtedly be more volatile than the Taiwanese storage sites. It is proposed that the merA+ D. radiodurans will be an effective step in the remediation of soil contaminated by a mixture of radioactive wastes by efficiently and effectively converting Hg(II) to Hg(0). The genetic manipulations designed by Brim et al. (2000) will be employed in this study. Brim and colleagues have successfully generated a number of strains of D. radiodurans that express the mercuric ion reductase that is responsible for the reduction of Hg(II) to Hg(0).

Experimental strategy/design

This project will serve as a pilot study to determine if genetically modified Deinococcus radiodurans can be effectively utilized to remediate waste soil in a controlled setting with the expectation that this model can be employed on a much larger scale in ground soils in areas surrounding Orchid Island and North Korean waste sites and perhaps in similar situations elsewhere. Due to the closed nature of the DPRK the first phase of the pilot will be conducted based on samples of waste and soil from the Orchid Island sites. Preliminary testing will include culturing D. radiodurans from Orchid Island soil samples. The effectiveness of the intervention will be assayed using fluorescent labeling via GFP fusions to the merA mutation.

Phase 1: Staff members will need to be selected in order to include an expert in each of the following: geology, ecology, extremophile biology, agriculture, physics, sociology of the region, as well as the Taiwanese and Korean languages. Taipower will be contacted in a collaborative manner, highlighting that this study or studies like it may pave the way for significant soil remediation both in their present site and in any future waste disposal sites. Taipower will be solicited for advice on handling the materials and get information about its contents. By establishing a good rapport with them we may be able to more easily get in contact with North Korean officials. Research headquarters will be located on Orchid Island. Contact must be made with the Yami, who may be interested in a project that will aid in the cleaning of the soil.

Phase 2: Indigenous D. radiodurans will be isolated and enumerated from soil on Orchid Island in order to give evidence for the safety of adding cultures to the soil. Soil samples will be taken and then irradiated at around 30Gy/hour in order to kill all non-radioresistant bacteria. MerA+ D. radiodurans strains will be created as per Brim et al (2000) with ampr in order to select for relevant cultures. An additional plasmid will be introduced that will contain Enhanced Green Fluorescent Protein and kanamyacin resistance with merA promoters. The expression of GFP will be an effective way of correlating the reduction of Hg to the expression of merA. The kanr will be used as a method of selecting for cells that have incorporated the desired manipulation. In addition, it will be important to maintain the balance of the niche by ensuring that modified populations do not out-compete indigenous ones. Thus, strains of D. radiodurans will be selected that have defects that will render them unable to compete with unmodified D. radiodurans in the absence of Hg(II). Strains will be selected for weakened heat stress response and weakened ability to deal with oxidative stress. Soil samples will be evaluated for levels of Hg(II) and Hg(0) as well as the intensity of the ionizing radiation per hour.

Phase 3: In a series of above ground soil tanks containing contaminated soil, merA+ D. radiodurans will be added. Soil will be carefully monitored for concentration of both states of Hg.

Phase 4: Based on results from phase 3, necessary follow up studies will be completed and results will be written up. There must be two copies of the report; one targeted to the scientific community and another targeted toward the Yami. It is very important that the Yami remain informed of the matter on an equal basis with Taipower. This will pose a unique challenge because Yami is a purely verbal language and has no written form. Thus, with the assistance of the Yami community, Yami individuals with some technical training will be hired to serve as a communications link between the study and the Yami community.

Legal and ethical issues

There are a number of possible ways to address the legitimate concern of releasing a genetically modified organism into nature. This study will ensure that the modified version of D. radiodurans will not out-compete the indigenous strains through the selection of strains that are weak in a number of phenotypes that are critical to the viability of the organism. Thus, the modified organisms will be balanced between surviving in situations of Hg contamination due to their competitive advantage from the merA manipulation, and being unable to out-compete the indigenous strains in uncontaminated situations. It is anticipated that either the modified strains will remediate in their immediate environment, then lose the position of advantage in the niche, or that after several generationseven in the presence of Hgthe selected weaknesses will cripple the ability for the strain to replicate. There are a number of ways that control of the modified population can be addressed via additional genetic manipulations, but the proposed method is preferred because those who protest genetically modified organisms in the wild will not accept another modification to hold the population in check. This proposed method will simply preferentially select from the indigenous bacterial population.

Applicability to other countries

There is great potential for applying this model to other countries that have industrial waste storage facilities. There are many thousands of radioactive waste storage facilities in countries throughout the world. In addition, D. radiodurans is a highly likely candidate for the remediation of a number of other types of waste situations because of its unique gene plasticity and its ability to replicate large portions of foreign DNA. D. radiodurans is a good model for the remediation of complex wastes because of the great potential for the insertion of gene cassettes. The future holds great potential for the application D. radiodurans in soil remediation efforts around the globe.

Acknowledgments

It is my pleasure to thank Professor Amy Vollmer for her guidance during all phases of this proposal. Thanks to Professors Murer and Nackenoff for personal communications with regard to the political climate in the DPRK. Thanks to Tammy Rabbideau for her assistance in the early stages of research. Also, thanks to Maryann Chambers and Stephanie Cross for their critiques of later drafts of the proposal.

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